The current MVA virus vaccine provides an extremely safe alternative to the currently available smallpox vaccines that are associated with a significant degree of adverse effects. However, replication of MVA virus is impaired in human cells and cells of many animal species, providing a natural limitation on infections with this virus. Thus, large doses of MVA are needed, and multiple doses are required for effective protection against smallpox and other agents. Methods to enhance the immunogenicity of this vaccine virus, preferably to the point of reducing the vaccination to a single dose, would significantly enhance the efficacy of this vector.
One of the unusual features of poxviruses is their ability to produce virus particles of more than one type. The majority of the orthopoxviruses can produce infectious virus particles of several types differing in either surface structure or site of accumulation. Most abundant, and often representing more than 90% of virus progeny, are the intracellular mature viruses (IMVs), which are generally thought to possess a single membrane. A small proportion of the IMV are converted to intracellular enveloped virus (IEVs), which are IMV wrapped in double membranes derived from the trans-Golgi network or tubular endosomes. The IEV are transported to the cell surface, where their outer membranes fuse with the plasma membrane to produce either the released, extracellular enveloped virus (EEV) or the cell-associated enveloped virus (CEV), which remains attached to the cell surface. Those IMVs that are not converted into IEV, CEV, or EEV, remain in the cytoplasm of the cell, either as free particles, or as particles embedded within A-type inclusions (ATIs). ATIs are large, well-defined proteinaceous bodies produced in cells infected with certain strains of cowpox, ectromelia, raccoonpox virus, fowlpox virus, or canarypox viruses (FIG. 1). The different kinds of virus particles have distinct physical, immunological, and biological properties, suggesting that particles of each type provide some advantages for virus replication, however, the in vivo roles of particles of each of these types are not fully understood.
The inclusion of IMV within the ATIs is determined by two factors, the synthesis of an ATI matrix protein, and the synthesis of the P4c protein, which directs the IMV into the ATIs (FIG. 1). Both the ati gene (Patel and Pickup, EMBO J. 6(12):3787-3794 (1987), Patel et al, Virology 149(2):174-89 (1986)), and the p4c gene (McKelvey et al, J. Virol. 76(22):11216-25 (2002)) have been identified. Interestingly, although few orthopoxviruses synthesize ATIs (because they encode truncated ATI proteins), most orthopoxviruses, including variola and monkeypox viruses, encode a P4c protein. Consequently, in orthopoxviruses of most types, including variola virus, the repair of a single gene, the ati gene, or the provision of the ATI protein by complementation, will result in the production of ATIs containing IMV.
Presumably, the virus-containing ATIs (V+ ATIs) facilitate the transmission of the virus particles from one host to another. The role of the ATI in virus dissemination and pathogenesis within the infected animal is unknown. The effects of ATIs upon immunity to the virus are also unknown. The IMVs within an ATI are expected to be inaccessible to antibodies that might otherwise neutralize these particles. Moreover, the properties of the ATIs suggest that the IMV within ATI particles are likely to be resistant to antibody neutralization of virus infectivity, whatever the nature of the antibodies (anti-IMV, EEV or ATI).
Currently, it is unclear how IMVs within ATIs might become able to infect host cells. Initial analyses of the fate of V+ATIs added to human 293 cells (FIG. 2) suggest that the ATIs can adhere to target cells, and then IMVs at the interface between the cell and the ATI are able to initiate an infection through the plasma membrane. Cell surface proteases may facilitate the degradation of ATI protein to release the IMV, while maintaining the IMV in a protected environment between the ATI and the cell surface. In this way, the ATI will release only a portion of its IMVs to infect one cell, while retaining the remainder to infect other cells. In effect, the ATI could act as a delivery vehicle to inoculate multiple IMVs into a number of different cells over a period of time, and at the same time protect the embedded IMVs from physical and immunological inactivation. This type of infection process might well protect the IMVs from complement-mediated attack or any neutralizing antibodies that might otherwise interfere with the process of IMV infection.
In addition to affecting the uptake of infectious virus, the two proteins required for V+ ATI formation are likely to be important targets for immune responses. The ATI protein is typically one of the most abundant proteins synthesized in orthopoxvirus infected cells, even if the virus, like vaccinia virus (FIG. 3) encodes a truncated protein (the 94 kDa ATI protein) incapable of forming discrete ATIs. The P4c protein is highly conserved among orthopoxviruses, including variola virus, and it is the largest IMV surface protein, and one of the most abundant IMV surface proteins (Katz and Moss Proc. Natl. Acad. Sci. USA 66(3):677-84 (1970), Katz and Moss, J. Virol. 6(6):717-26 (1970), Sarov and Joklik, Virology 50(2):579-92 (1972)) (FIG. 4). Two early studies (Oie and Ichihashi, Virology 157(2):449-59 (1987), Stern and Dales, Virology 75(1):232-41 (1976)) suggested that the P4c protein is a target for antibodies capable of neutralizing IMV, although Dales later reported an inability to confirm this (Wilton et al, Virology 214(2):503-11 (1995)).
The present invention relates to MVA virus vaccine modified by repair of two inactivated viral genes to forms that embed the infectious virus in ATI particles. These vaccines have greater utility and efficacy than current MVA vaccine viruses because of enhanced particle stability, enhanced deliverability by nasal, dermal, intramuscular or oral routes, reduced release of infectious virus and enhanced immunogenicity.